Biology subjects

Colgan, S. P.

Publications and source records attributed to Colgan, S. P..

9 recordsLinked to original sources

bioRxiv2026

Mitochondrial ATP production promotes T cell differentiation and function by regulating chromatin accessibility

Immune elimination of chronic infection or cancer requires cytotoxic CD8+ T cells that adopt and maintain an effector phenotype. Cytotoxic T cell function is a bioenergetically demanding process and T cells subjected to chronic antigen exposure have compromised effector function despite high rates of glycolysis. Here we report the ability of the short-chain -hydroxy acid, D--hydroxybutyrate, to act as a signaling molecule that increases mitochondrial ATP production and drives the conversion of proliferating T cells into cytotoxic effector cells. DAHB signaling switches ATP production from glycolysis to oxidative phosphorylation supported by fatty acid oxidation, even in glucose-replete media. This conversion suppresses both AMPK phosphorylation and the integrated stress response (ISR) in activated T cells while significantly elevating the level of the phosphagen, phosphocreatine (PCr). Both the PCr bioenergetic reserve and oxidative phosphorylation were required for T cell effector differentiation. DAHB-induction of CD8-effector gene transcription was coupled to bioenergetics by enhanced ATP-dependent remodeling of chromatin accessibility at effector gene loci. DAHB enhanced CD8+ T cell antitumor activity both in vitro and in vivo, and DAHB treatment of transferred T cells led to persistent in vivo antitumor effects. Together, these findings link cellular bioenergetics to the regulation of chromatin accessibility and gene expression required to support effector function.

immunology↗

bioRxiv2026

Metabolite mimicry identifies butyrate analogs with select protective functions in the intestinal mucosa

Microbial-derived short-chain fatty acids regulate a variety of pathways in the healthy colonic mucosa. In particular, butyrate serves as the primary energy source for colonocytes and regulates gene transcription by stabilizing the transcription factor hypoxia-inducible-factors (HIF) and functioning as a histone deacetylase (HDAC) inhibitor. A limitation of butyrate as a therapeutic is its rapid metabolism in differentiated colonocytes. Furthermore, intestinal stem cells (ISCs) respond differently to butyrate, preferentially using glucose for energy procurement. To address these limitations, we explored metabolite-mimicry to discover compounds with potent or selective biological responses within the butyrate pathway(s). We discovered an analog, 3-chlorobutyrate (3-Cl BA), that significantly enhances epithelial barrier formation and wound healing in vitro. Mechanistically, we revealed that 3-Cl BA is a potent HDAC inhibitor. Furthermore, unlike butyrate, 3-Cl BA does not stabilize HIF and it is not used as metabolic fuel. In vivo studies in a DSS-colitis model revealed that contrary to butyrate, 3-Cl BA is protective. Studies in stem-like colonoids demonstrated that only butyrate inhibits ISC proliferation and differentiation. Furthermore, it was recently reported that HIF stabilization inhibits ISCs activity. Given the fact that butyrate but not 3-Cl BA stabilizes HIF, we surmised that 3-Cl BA would circumvent these detrimental functional consequences. We demonstrate here that pharmacologic HIF stabilization inhibits colonoid differentiation and that genetic loss of HIF significantly promotes ISC differentiation. This study reveals a promising butyrate analog protective in colitis and demonstrates the advantages of metabolite-mimicry to dissect selective biological functions from major metabolites in the gut. Significance statementButyrate is a well-studied microbial short-chain fatty acid that regulates a number of mucosal pathways and is paramount in maintaining intestinal integrity. In health, it is a major source of energy for colonocytes and regulates gene transcription. The role of butyrate in disease is still controversial and not well understood. When butyrate is not metabolized or well-utilized (e.g. disease), it accumulates in intestinal stem cells leading to reduced cell proliferation and differentiation, thereby hampering intestinal barrier recovery. In this study, we describe a butyrate analog that enhances epithelial barrier formation and wound healing. Furthermore, as opposed to native butyrate, this butyrate analog is protective in a colitis mouse model and does not exhibit detrimental influences on intestinal stem cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/697087v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1dd0f0borg.highwire.dtl.DTLVardef@6d27forg.highwire.dtl.DTLVardef@1e5d07corg.highwire.dtl.DTLVardef@dab74f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Microbially derived butyrate plays a key role in intestinal homeostasis. It is the primary source of energy for colonocytes, contributing to a metabolic and oxygen gradient as it is metabolized by differentiated cells along the intestinal crypt. Through the regulation of transcription factors such as HIF and the inhibition of HDAC, it regulates barrier formation and wound healing promoting a strong tight junction profile. Furthermore, well oxygenated ISCs at the bottom of the crypt are unaccustomed to the effects of butyrate, including HIF stabilization (left). In disease, loss of intestinal architecture leads to a disrupted metabolic/oxygen gradient where butyrate accumulates in stem cells leading to decreased proliferation, differentiation, and increases in apoptosis. 3-Cl BA selectively acts as an HDACi and does not stabilize HIF, exhibiting no significant detrimental effects on ISCs (right). Created in BioRender. Ornelas, A. (2026) https://BioRender.com/x9cy8mw C_FIG

molecular biology↗

bioRxiv2025

Proton motive force as a driver of persister sensitization by adenosine

Antibiotic tolerance and persistence contribute to the emergence of antimicrobial resistance, yet strategies to reverse these phenotypes remain limited. Our previous work revealed that the naturally occurring nucleoside adenosine can reverse antibiotic tolerance in diverse bacterial strains by modulating cellular energetics. Here, we define the mechanism underlying this potentiation, identifying adenosine metabolism as a driver of cytoplasmic alkalinization and proton motive force (PMF) generation. Using RNA sequencing, metabolite assays, and pH-sensitive fluorescent reporters, we show that adenosine is catabolized by purine nucleoside phosphorylase (deoD) to yield ribose-1-phosphate, which enters the pentose phosphate pathway. This metabolic flux stimulates the electron transport chain, leading to proton translocation, increased cytoplasmic pH, and enhanced PMF. Disruption of key metabolic enzymes (deoD, deoB, tktAB) or inhibition of enolase abolishes both alkalinization and antibiotic sensitization. Using a respiratory-deficient mutant, we demonstrate that aerobic respiration is the primary driver of alkalinization and gentamicin potentiation, though adenosine can partially increase membrane potential independently of oxidative phosphorylation. These findings support a model in which adenosine metabolism promotes aminoglycoside uptake via PMF-driven transport, sensitizing tolerant bacteria to killing. Our work implicates the ribose moiety of adenosine as a key metabolic lever for reversing tolerance. Broader exploration of nucleoside-based adjuvants across bacterial species and antibiotic classes may reveal generalizable strategies to enhance antibiotic efficacy against recalcitrant infections.

microbiology↗

bioRxiv2025

Regulation of Epithelial HIF by Probiotic Escherichia coli

The gastrointestinal tract is home to trillions of microorganisms that interact with their host in profound ways, including regulation of immune, endocrine, and neurological functions. One mechanism by which these microbes interact with their eukaryotic host is through the generation of short-chain fatty acids (SCFAs), which are metabolized by the intestinal epithelium creating a state of "physiologic hypoxia". This hypoxia, in turn, results in stabilization and activation of hypoxia-inducible factor (HIF), a transcription factor family shown to support gut barrier function and homeostasis, in the intestinal epithelium. The association between HIF and intestinal homeostasis has been long understood, as both genetic and pharmacologic potentiation of the HIF signaling pathway has been shown to promote barrier function both in vitro and in vivo. Although it has been previously established that pathogenic bacteria regulate HIF stabilization and activity in the intestinal epithelium independent of SCFA metabolism, it is not clear whether this property extends to noninfectious and/or commensal bacterial species. Here, we demonstrate that nonpathogenic, commensal strains of Escherichia coli stabilize HIF in intestinal epithelial cells in vitro. Further, we show that HIF is transcriptionally active in these cells and drives a "pro-barrier" transcriptional program. This property was found to be dependent on bacterial aerobic respiration, as genetic elimination of E. coli aerobic respiration abolished HIF stabilization and the subsequent transcriptional phenotype. Finally, we observed induction of tissue hypoxia in vivo using antibiotic-treated mice colonized with wild-type, but not respiration-deficient, E. coli. These findings demonstrate a novel ability for probiotic E. coli to regulate intestinal homeostasis through activation of HIF and suggest that this mechanism might be leveraged in as a novel therapeutic to combat intestinal inflammation, such as that observed during inflammatory bowel disease (IBD).

cell biology↗

bioRxiv2024

Epithelial heme oxygenase-1 enhances colonic tumorigenesis by inhibiting ferroptosis

Induction of heme oxygenase-1 (HO-1/Hmox1) is broadly considered cytoprotective, but the role of colonic epithelial HO-1 in colitis-associated tumorigenesis is poorly defined. HO-1 catabolizes heme, releasing ferrous iron, a key driver of oxidative stress and lipid peroxidation. We observed that colonic epithelial HO-1 is induced during colitis and tumorigenesis. We also found that HO-1 is upregulated in ferroptosis-inducing conditions in murine and human colonic epithelial organoids, and correlated with lipid peroxidation and ferroptosis markers in colonic tumors. In colonic epithelial organoids exposed to heme, deletion of Hmox1 amplified a compensatory oxidative stress and detoxification transcriptional program, likely reflecting unresolved oxidative and non-oxidative toxicity from heme. In vivo, epithelial HO-1 deficient mice developed significantly fewer and smaller tumors compared to littermate controls in a colitis-associated tumorigenesis model, despite similar inflammatory injury. Tumors from knockout mice exhibited reduced iron levels, decreased lipid peroxidation, lower oxidative DNA damage, and decreased proliferation. Single-cell RNA sequencing of tumor epithelial cells revealed a shift from a proliferative to a stress-adaptive program with loss of HO-1. These findings identify epithelial HO-1 as a context-dependent regulator of tumorigenesis: protective against acute heme toxicity, but promoting iron-dependent oxidative damage and proliferation in the setting of chronic inflammation.

cancer biology↗

bioRxiv2023

Fundamental role for the creatine kinase pathway in protection from murine colitis

Inflammatory diseases of the digestive tract, including inflammatory bowel disease (IBD), cause metabolic stress within mucosal tissue. Creatine is a key energetic regulator. We previously reported a loss of creatine kinases (CKs) and the creatine transporter expression in IBD patient intestinal biopsy samples and that creatine supplementation was protective in a dextran sulfate sodium (DSS) colitis mouse model. In the present studies, we evaluated the role of CK loss in active inflammation using the DSS colitis model. Mice lacking expression of CKB/CKMit (CKdKO) showed increased susceptibility to DSS colitis (weight loss, disease activity, permeability, colon length and histology). In a broad cytokine profiling, CKdKO mice expressed near absent IFN-{gamma} levels. We identified losses in IFN-{gamma} production from CD4+ and CD8+ T cells isolated from CKdKO mice. Addback of IFN-{gamma} during DSS treatment resulted in partial protection for CKdKO mice. We identified basal stabilization of the transcription factor hypoxia-inducible factor (HIF) in CKdKO splenocytes and pharmacological stabilization of HIF resulted in reduced IFN-{gamma} production by control splenocytes. Thus, the loss of IFN-{gamma} production by CD4+ and CD8+ T cells in CKdKO mice resulted in increased colitis susceptibility and indicates that CK is protective in active mucosal inflammation.

immunology↗

bioRxiv2023

Synaptopodin is necessary for Shigella flexneri intercellular spread

For many intracellular pathogens, their virulence depends on an ability to spread between cells of an epithelial layer. For intercellular spread to occur, these pathogens deform the plasma membrane into a protrusion structure that is engulfed by the neighboring cell. Although the polymerization of actin is essential for spread, how these pathogens manipulate the actin cytoskeleton in a manner that enables protrusion formation is still incompletely understood. Here, we show that butyrate responsive pathways promote intercellular spread by Shigella flexneri. We identify the mammalian actin binding protein synaptopodin, a butyrate responsive gene, as required for efficient intercellular spread of S. flexneri and Listeria monocytogenes. We show synaptopodin enhances the recruitment of actin to bacteria and stabilizes the actin tail. We show that, for S. flexneri, synaptopodin presence enables protrusions to form and to resolve at a greater rate, indicating that greater stability of the actin tail enables the bacteria to push against the membrane with greater force. We demonstrate that synaptopodin recruitment around bacteria requires the bacterial protein IcsA, and we show that this recruitment is further enhanced in a type 3 secretion system dependent manner. These data establish synaptopodin as required for intracellular bacteria to stabilize the actin cytoskeleton in a manner that enables efficient protrusion formation and identify for the first time that synaptopodin contributes to bacterial pathogenesis.

microbiology↗

bioRxiv2023

The TNF{blacktriangleup}ARE mouse as a model of intestinal fibrosis

Background & AimsCrohns disease (CD) is a highly morbid chronic inflammatory disease. The majority of CD patients also develop fibrostenosing complications. Despite this, there are no medical therapies for intestinal fibrosis. This is in part due to lack of high-fidelity biomimetic models to enhance understanding and drug development. There is a need to develop in vivo models of inflammatory bowel disease-related intestinal fibrosis. We sought to determine if the TNF{Delta}ARE mouse, a model of ileal inflammation, may also develop intestinal fibrosis. MethodsSeveral clinically relevant outcomes were studied including features of structural fibrosis, histological fibrosis, and gene expression. These include the use of a luminal casting technique we developed, traditional histological outcomes, use of second harmonic imaging, and quantitative PCR. These features were studied in aged TNF{Delta}ARE mice as well as in cohorts of numerous ages. ResultsAt ages of 24+ weeks, TNF{Delta}ARE mice develop structural, histological, and genetic changes of ileal fibrosis. Genetic expression profiles have changes as early as six weeks, followed by histological changes occurring as early as 14-15 weeks, and overt structural fibrosis delayed until after 24 weeks. DiscussionThe TNF{Delta}ARE mouse is a viable and highly tractable model of intestinal fibrosis. This model and the techniques employed can be leveraged for both mechanistic studies and therapeutic development for the treatment of intestinal fibrosis.

cell biology↗

bioRxiv2019

Endothelial specific PER2 at the crossroads of light elicited circadian amplitude enhancement as novel cardioprotective strategy and transcriptional regulation of HIF1A-dependent metabolic adaptation to myocardial ischemia

Consistent daylight oscillations and abundant oxygen availability are fundamental to human health. While both are connected from an evolutionary and cellular perspective, only oxygen is an established therapy in cardiovascular medicine. Here, we probe the mechanistic intersection between light-(Period 2, PER2) and oxygen-(hypoxia inducible factor, HIF1A) sensing pathways in cellular adaptation to low oxygen conditions with respect to myocardial ischemia. Using a whole genome array from daylight exposed wildtype or Per2-/- mice, an affinity purification-mass spectrometry-based proteomics screen for PER2 targets in hypoxic human endothelial cells, and targeted metabolomics from human healthy volunteers after daylight therapy, we investigated the intersection of light and hypoxia elicited pathways. Housing mice under daylight conditions prior to myocardial ischemia and reperfusion (IR)-injury, uncovered circadian PER2 amplitude enhancement as novel cardioprotective strategy, mimicking HIF1A metabolic adaptation to myocardial ischemia in a PER2 regulated manner. Whole genome array analysis from daylight exposed wildtype and Per2-/- mice or myocardial IR-injury in endothelial specific PER2 deficient mice (Per2loxP/loxP-VE-Cadherin -Cre) revealed a critical role for light elicited PER2 in maintaining the endothelial barrier function during myocardial ischemia. Mechanistic studies in human endothelia pointed towards a master transcriptional regulatory role for endothelial PER2 in metabolic reprograming to hypoxia via HIF1A, which was mimicked during normoxic PER2 stabilization. Translational investigation of light elicited pathways in human healthy volunteers found similar increases of PER2 or mimicking of HIF1A dependent metabolism. These studies identify light elicited circadian amplitude enhancement of endothelial PER2 as a novel cardioprotective strategy. Furthermore, these studies identify PER2 as critical control point of endothelial metabolic reprograming to maintain vascular integrity during myocardial IR-injury and implicate the use of daylight therapy to increase endothelial PER2 signaling as a strategy for the treatment of coronary artery disease.

cell biology↗